Conventionally, in the clinical diagnosis of the infection disease due to viruses, bacteria or the like, identification of a pathogen such as viruses and bacteria has been carried out by culturing an analyte using body fluid such as blood, feces and sputum as a test sample. However, these methods have such problems that very long period of time is required to culture the analyte and culture itself dose not go smoothly in some cases of viruses or bacteria. Further, considering that a special technique is required to culture such analytes, they are not necessarily good methods to obtain a satisfactory result rapidly and simply.
A method, in which the disease agent such as a virus or bacterium is identified by an antibody-antigen reaction, has been also performed. It is a good method in view of rapidness and simplicity, since automatic analysis is possible. However, in an antibody detection method for detecting a disease agent as an antibody, there is a problem in sensitivity since sometimes lack of volume of the disease agent in a test sample results in impossibility of detecting the disease agent. Further, there is a problem that it is difficult to decide an antibody site specific to a kind of the disease agent. On the other hand, as for an antibody detection method for detecting an antibody produced in a body caused by infection of a disease agent, since some period of time is necessary from the infection with a disease agent to the production of the antibody, there is a problem that the disease agent can not be detected during the period of time.
On the contrary, a method for detecting a nucleic acid fragment having a specific base sequence in accordance with the kind of viruses or bacteria (a target nucleic acid fragment) by utilizing complementarity of the base sequence is the method that allows a direct identification of the disease agent. Therefore, it has come into wide use as a gene examination method such as a DNA probe method or a PCR (polymerase chain reaction) method. For example, the method for examining the HCV (hepatitis C virus) gene displays its greatest force in an inspection of the interferon (INF) administration or monitoring of cure in INF remedy fop hepatitis C, since the volume of HCV is directly known by the method.
It is further expected that genotype of respective disease agents such as viruses or bacteria will be elucidated, and that new curative medicines will be developed utilizing the genotype. In this case, it is very important not only to identify a disease agent but also to know genotype of the disease agent. The gene examination method is really the method that meets the request.
Furthermore, since the gene examination method can directly detect personal gene characteristics, in addition to identification of disease agent, it can be used to detect mutation of a gene that is the cause of hereditary disease or to detect a gene factor that affects aptitude of one's liability to life-style related to diseases such as cancer or diabetes. Especially, when the whole base sequences will be determined, it is expected that, as the post genome research, the relation between gene characteristics and disorder will be solved in greater detail, and that curative medicines using the gene characteristics will be developed. It is forecasted that demand for the gene examination method will continue to increase along with the progress of the post genome research.
However, since the gene examination methods preformed these days require a special technique, complex operation, special device and the like, only large scale examination centers can perform the gene examination method. In the examination of either infection disease caused by a virus, bacterium or the like or personal gene characteristics, an on the spot and quickest decision of guideline for diagnosis and remedy exerts greater effectiveness. In order to realize this situation, it is vital to provide a new gene examination method that can be operated by any operator with easiness and can give the examination result promptly.
For the purpose of improving simplicity and promptness, such gene examination methods have been developed that utilize detection of progress of a polymerase extension reaction using a target nucleic acid as a template. A method for detecting a generation process of an amplified product in real time as alteration of fluorescence strength during amplification of a specific nucleic acid region of a target nucleic acid fragment by PCR (Real Time PCR method) is a good method in terms of promptness since it does not require steps of electrophoresis of the amplified product after PCR and analysis of its result. Therefore this method is commercialized as the “TAQMAN” probe method (PE Biosystems Co.) or the Molecular Bean method (Stratagene Co.). However the methods utilize FRET (fluorescence resonance energy transfer), and have a problem that they require, on operation, a device that can measure alteration of fluorescence intensity and preparation of a specific hybridization probe in which a fluorescent dye and a quencher are combined to be labeled. Thus, they are no better than a special technique.
A method is described in “Igaku no Ayumi (Progress of Medical Science)” Vol. 173, No 12, 1995, in which alteration of fluorescence strength is detected while amplifying a specific nucleic acid region of a target nucleic acid under presence of fluorescent substance having a intercalator characteristics (IM-PCR: intercalation monitoring PCR method). This method has an advantage as the Real Time PCR method to the extent that it does not require a specific hybridization probe. However, the method also requires a device on operation that can measure the alteration of fluorescence strength. Further, the method has a problem in specificity since the fluorescent substance having intercalator characteristics connects to the all nucleic acid fragments existing in the system despite of presence or absence of PCR amplification of the specific nucleic acid region of the target nucleic acid fragment.
On the other hand, examples, in which amplification of DNA by PCR is detected as an electrochemical response by utilizing an intercalator, are disclosed in “2000 Electrochemistry Autumn Meeting, Preprint 2F17” and “2001 Electrochemistry Autumn Meeting, Preprint 1K11”. These methods are good in terms of simplicity and a little space since they do not require measurement of fluorescence intensity alteration. However, there still remains a problem, on operation, in detection sensitivity and reproducibility of measurement, since the intercalator used is not a threading intercalator having redox activity and detection of DNA amplification by PCR is performed by detecting decrease of the electrochemical response. On this point, these methods are different from a method for analyzing a nucleic acid fragment according to the present invention, in which detection of a double-stranded nucleic acid fragment formed by polymerase chain reaction utilizing a target nucleic acid fragment as a template is performed by measuring increase of an electrochemical response under the presence of a threading intercalator having a redox activity.